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Purification and concentration of RNA polymerase on Ni-lipid monolayers

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Abstract

Knowledge of the chromatin transcription intermediate structures can shed light on the functioning of RNA polymerase in the cell, which is important both for basic science and for the development of therapeutic approaches for the treatment of diseases associated with transcription disorders. In this study, we used affinity monolayers formed by lipids bound to Ni to concentrate RNA polymerase from a dilute solution containing glycerol. It has been shown that RNA polymerase can be isolated and concentrated on a Ni-lipid monolayer from solutions containing cryoprotectants, which usually make it difficult to visualize samples by electron microscopy. This method of isolation allowed us to obtain more diverse spatial orientations of molecules on carbon film, which facilitates three-dimensional reconstruction from TEM data. The results obtained can be used in further structural studies of the transcription processes to identify interactions in transcriptional complexes containing RNA polymerase.

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Abbreviations

TEM:

transmission electron microscopy

EC:

elongation complex

DGS-NTA (Ni):

1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt)

PMSF:

phenylmethylsulfonyl fluoride

EDTA:

sodium ethylenediaminetetraacetate

SDS-PAAG:

denaturing polyacrylamide gel with sodium dodecyl sulfate

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Authors and Affiliations

  1. Department of Bioengineering, School of Biology, Moscow State University, Moscow, 119234, Russia

    M. G. Karlova, O. I. Volokh, O. V. Chertkov, M. P. Kirpichnikov, V. M. Studitsky & O. S. Sokolova

  2. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117437, Russia

    M. P. Kirpichnikov

  3. Fox Chase Cancer Center, Philadelphia, PA, 19111, USA

    V. M. Studitsky

Authors
  1. M. G. Karlova
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  2. O. I. Volokh
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  3. O. V. Chertkov
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  4. M. P. Kirpichnikov
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  5. V. M. Studitsky
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  6. O. S. Sokolova
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Corresponding author

Correspondence to O. S. Sokolova.

Additional information

Original Russian Text © M.G. Karlova, O.I. Volokh, O.V. Chertkov, M.P. Kirpichnikov, V.M. Studitsky, O.S. Sokolova, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 6, pp. 637–643.

The article is published based on the information presented at the VIII Russian Symposium “Proteins and Peptides,” September 18–22, 2017, Moscow.

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Karlova, M.G., Volokh, O.I., Chertkov, O.V. et al. Purification and concentration of RNA polymerase on Ni-lipid monolayers. Russ J Bioorg Chem 43, 658–663 (2017). https://doi.org/10.1134/S1068162017060048

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  • DOI: https://doi.org/10.1134/S1068162017060048

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